Positional delivery and encoding by oligonucleotides of biological cells for single cell sequencing (POS SEQ)

ABSTRACT

Techniques for positional delivery and position encoding by oligonucleotides of biological cells for single cell RNA sequencing are provided. In one aspect, a method of positional delivery and encoding of cells in a biological sample includes: encoding the cells in the biological sample for single cell sequencing by delivering molecular probes inside the cells that encode a position of the cells in the biological sample. A system for positional delivery and encoding of cells in a biological sample is also provided.

FIELD OF THE INVENTION

The present invention relates to single cell sequencing, and moreparticularly, to techniques for positional delivery and positionencoding by oligonucleotides of biological cells for single cellribonucleic acid (RNA) sequencing, i.e., positional sequencing (POSSEQ).

BACKGROUND OF THE INVENTION

The successful functioning of multi-cellular organisms relies on thecoordinated functions of a multitude of molecular constituents fromindividual cells and the interactions among functionally distinct cells.Further, these molecular constituents are constantly changing such as inresponse to cell-to-cell interactions which oftentimes result from localphysical cell-to-cell contact and/or from short length-scale paracrinecell-to-cell communications. Thus, the state of a biological system isoften defined by the relative position of the cells in the system andthe highly dimensional molecular composition of each of those cells.

For example, with diseases such as cancer, specific tumor cellsubpopulations can co-opt adjacent normal cells to support tumorprogression. Thus, the relevance of cell positioning has motivated thedevelopment of therapeutic agents that target the co-opted cells, suchas platelet-derived growth factor receptor (“PDGFR”) inhibitors totarget PDGFR+ pericytes, and small molecule inhibitors or neutralizingantibodies of colony-stimulating factor 1 (“CSF1”) receptors to targetmacrophages.

Typically, spatial and molecular measurements are made using image-basedanalysis where molecular and positional information is obtained bytaking microscopy images of samples treated with either enzymatically-or fluorescently-labeled antibodies that bind specifically to themolecular target of interest. When the images are digital, the sensorpixel position reflects the spatial relationship of the cells, while thesensor pixel signal intensity reflects the local density of the labeledantibodies molecular target of interest.

Other techniques employed for concomitant spatial and molecularmeasurements involve first recording the positioning of the individualcells that are then measured. It is however impractical to implementsuch a technique with potentially millions of distinct cells that needto be stored and processed separately for molecular profiling.

Thus, improved techniques for concomitant spatial and molecularmeasurements of biological cells would be desirable.

SUMMARY OF THE INVENTION

The present invention provides techniques for positional delivery andposition encoding by oligonucleotides of biological cells for singlecell ribonucleic acid (RNA) sequencing (POS SEQ). In one aspect of theinvention, a method of positional delivery and encoding of cells in abiological sample is provided. The method includes: encoding the cellsin the biological sample for single cell sequencing by deliveringmolecular probes inside the cells that encode a position of the cells inthe biological sample.

In another aspect of the invention, another method of positionaldelivery and encoding of cells in a biological sample is provided. Themethod includes: constructing a cDNA library of molecular probes thatencode a position of cells in a biological sample; linking the molecularprobes to a vessel; delivering the vessel with the molecular probes tospecific locations of the biological sample where the vessel deliversthe molecular probes inside the cells at the specific locations;extracting the cells containing the molecular probes from the sample;and performing single cell sequencing of the extracted cells.

In yet another aspect of the invention, a system for positional deliveryand encoding of cells in a biological sample is provided. The systemincludes: a processor device, connected to a memory, that is implementedto: analyze data from single cell sequencing of cells along withmolecular probes, that have been delivered inside the cells, whichuniquely encode a position of the cells in a biological sample.

A more complete understanding of the present invention, as well asfurther features and advantages of the present invention, will beobtained by reference to the following detailed description anddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating an exemplary methodology of positionaldelivery and encoding of cells in a biological sample for single cellsequencing according to an embodiment of the present invention;

FIG. 2 is a diagram illustrating an exemplary barcoded deoxyribonucleicacid (DNA) oligonucleotide primer molecule according to an embodiment ofthe present invention;

FIG. 3 is a diagram illustrating an exemplary methodology forconstructing a cDNA library using Moloney murine leukemia virus reversetranscriptase (MMLV RT) technology according to an embodiment of thepresent invention;

FIG. 4 is a diagram illustrating an exemplary methodology for a‘template switch’ by the MMLV RT using a template switch oligonucleotide(TSO) sequence according to an embodiment of the present invention;

FIG. 5 is a diagram illustrating use of a lentiviruses as the vessel fordelivering a location-specific molecular probe inside the cells of abiological sample according to an embodiment of the present invention;

FIG. 6 is a diagram illustrating use of a disulfide-linkedcell-penetrating peptide (CPP) as the vessel for delivering alocation-specific molecular probe inside the cells of a biologicalsample according to an embodiment of the present invention;

FIG. 7 is a diagram illustrating use of bead micro-particles as thevessel for delivering a location-specific molecular probe inside thecells of a biological sample according to an embodiment of the presentinvention;

FIG. 8 is a diagram illustrating an exemplary methodology for using aliquid cargo delivery device to deliver the vessels with uniquemolecular probes to specific locations of the biological sample, wherethe vessel delivers the location-specific molecular probes inside thecells at those locations for single cell sequencing according to anembodiment of the present invention;

FIG. 9 is a diagram illustrating an exemplary apparatus for performingone or more of the methodologies presented herein according to anembodiment of the present invention;

FIG. 10 depicts a cloud computing environment according to an embodimentof the present invention; and

FIG. 11 depicts abstraction model layers according to an embodiment ofthe present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Provided herein are techniques for concomitant positional and molecularmeasuring of cells using a molecular probe having a uniqueoligonucleotide sequence that encodes a positioning of the cells withina biological sample such as a cell culture (e.g., a cell cultureincluding living eukaryotic and/or prokaryotic cells) and/or a tissuesample (e.g., a biopsy, formalin-fixed paraffin-embedded (“FFPE”) and/orfrozen tissue containing living cells). Thus, when the cells are laterdissociated from the biological sample and sequenced, the cells willtake with them the positional information encoded in the molecularprobe. Advantageously, the molecular probe encodes the position at whicheach of the cells being sequenced is located within the biologicalsample.

As will be described in detail below, the molecular probe is firstlinked to a vessel such as a retrovirus, disulfide-linkedcell-penetrating peptide (CPP) and/or bead micro-particle. A liquidcargo delivery device such as a microfluidic probe (MFP) is then used todeliver the molecular probe/vessel to specific locations of thebiological sample. See, for example, Juncker et al., “Multipurposemicrofluidic probe,” Nature Materials, Advanced Online Publication (July2005) (8 total pages), the contents of which are incorporated byreference as if fully set forth herein. By way of the vessel, themolecular probes with unique nucleotide sequences are delivered insidethe cells at those specific locations of the biological sample. Ashighlighted above, these oligonucleotide sequences are in effect a labelof the position of a given cell in the biological sample. Thus, for eachlocation (x,y) of a biological sample that the liquid cargo deliverydevice visits, a unique oligonucleotide sequence is delivered inside thecells at that location in the biological sample.

An overview of the present techniques for positional delivery andencoding of cells in a biological sample for single cell sequencing isnow provided by way of reference to methodology 100 of FIG. 1 . In step102, a complementary deoxyribonucleic acid (cDNA) library of molecularprobes containing unique oligonucleotide sequences is constructed. Aswill be described in detail below, according to an exemplary embodiment,the library construction leverages the template-switching activity ofMoloney murine leukemia virus reverse transcriptase (“MMLV RT”). For ageneral description of MMLV RT for library construction see, forexample, Zhu et al., “Reverse Transcriptase Template Switching: A SMART™Approach for Full-Length cDNA Library Construction,” BioTechniques30:892-897 (April 2001) (hereinafter “Zhu”), the contents of which areincorporated by reference as if fully set forth herein.

In step 104, the molecular probes with unique oligonucleotide sequencesare then linked to a particular vessel such as a retrovirus, coupled toa disulfide-linked cell-penetrating peptide (CPP) or a beadmicro-particle. This vessel will enable the molecular probes to bedelivered inside the cells of a biological sample. By delivering themolecular probes into the cells, the cells can be uniquelyidentified—even when disassociated from the biological sample—due to theunique oligonucleotide sequences carried by the molecular probes.

In step 106, the vessels with the molecular probes are delivered tospecific locations of the biological sample (e.g., a living cell cultureand/or tissue sample with living cells), where the vessels deliver themolecular probes inside the cells at those specific locations. Accordingto an exemplary embodiment, this location-specific delivery isaccomplished using a liquid cargo delivery device such a microfluidicprobe or MFP. A microfluidic probe is a non-contact, scanning platformthat can hydrodynamically localize as little as 100 picoliters of aliquid cargo with micrometer precision. For instance, the molecularprobes can be dispersed in a processing solution (e.g., an aqueoussolution) that is then delivered via the liquid cargo delivery device tospecific locations of the biological sample. The molecular probesdelivered to a given specific location of the biological sample containa unique oligonucleotide sequence that is associated with that givenspecific location. Thus, as provided above, when the cells are laterdisassociated from the biological sample for sequencing, theoligonucleotide sequence encodes the original position of the cells inthe sample (i.e., positional encoding).

Once the molecular probes are delivered to a given specific location ofthe biological sample, the vessels deliver the molecular probes insidethe cells at those specific locations. According to an exemplaryembodiment, the cells take in the vessels with the molecular probesthrough an active transfection/transduction process using living cellmachinery. Thus, the present techniques are preferably performed with aliving biological system. For instance, the biological sample preferablycontains living cells, whether as a living cell culture or as a tissuesample containing living cells. The living cells permittransfection/transduction to occur. Following the present positionalencoding process, the cells/tissue can be fixed if so desired.

In step 108, single cell sequencing is performed on the cells extractedfrom the biological sample. Even though the cells are disassociated fromthe biological sample for sequencing, the cells now contain themolecular probe with oligonucleotide sequence encoding the position ofthe cells in the biological sample. Thus, this positional data can beretained through the sequencing process.

For instance, in step 110 the data from the single cell sequencing isstored and analyzed (e.g., in silico) along with the data from themolecular probes which uniquely encodes the positions of the cells inthe biological sample. An exemplary apparatus for storing and analyzingthis data is provided in FIG. 9 , described below. Being able to analyzetranscriptomic data (i.e., RNA transcripts produced by a genome) fromthe cells along with the position of those cells in the biologicalsample is extremely beneficial. For instance, as highlighted above, thestate of a biological system is often defined by the relative positionof the cells in the system and the highly dimensional molecularcomposition of each of those cells. Take, for example, the developmentof therapeutic agents for cancer treatment that leverage cellpositioning to target specific tumor cell subpopulations. See above.

As described in conjunction with the description of step 102 ofmethodology 100 above, the process begins with the construction of acDNA library of molecular probes containing unique oligonucleotidesequences for positional encoding. As shown in FIG. 2 , the cDNA libraryconstruction begins with many barcoded DNA oligonucleotide primermolecules 204 conjugated to a microparticle 202 (e.g., a bead) such as aglass bead. The techniques for preparing distinctly barcodedoligonucleotide primers are described generally in Macosko et al.,“Highly Parallel Genome-wide Expression Profiling of Individual CellsUsing Nanoliter Droplets,” Cell 161, 1202-1214 (May 2015), the contentsof which are incorporated by reference as if fully set forth herein.

As shown in FIG. 2 , beginning from its 5′ end, each DNA oligonucleotideprimer molecule 204 contains a universal polymerase chain reaction (PCR)handle 204 a, a cell barcode 204 b, a unique molecular identifier (UMI)204 c, a position code 204 d, and a poly T sequence 204 e (i.e., Tn—asequence of n thymine repeats). PCR handle 204 a enables PCRamplification. For example, according to an exemplary embodiment, PCRhandle 204 a is a DNA oligonucleotide sequence for PCR primers in theamplification step (see, e.g., FIG. 4 —described below).

Cell barcode 204 b is a DNA oligonucleotide sequence that is unique tobead 202/cell into which the molecular probe is delivered. UMI 204 c isa DNA oligonucleotide sequence that is unique to this particular DNAoligonucleotide primer molecule 204. For instance, the DNAoligonucleotide primer molecules attached to the same bead 202 can sharethe same cell barcode, but different UMIs. In other words, the UMIs ofeach DNA oligonucleotide primer molecules has a different, uniqueoligonucleotide sequence. By way of example only, the UMIs can be usedfor normalizing gene counts during computational data processing. Forexample, the UMIs can be used to identify PCR duplicates during thesingle cell sequencing (see below).

The position code 204 d provides the (location-specific) oligonucleotidesequence for positional encoding. Namely, as described above, theposition code 204 d uses a unique oligonucleotide sequence to encode thelocation (x,y) of cells in a biological sample into which the presentmolecular probes will be delivered. A length of the position code 204 dcan depend on the total number of locations (x,y) to be encoded. Forexample, according to an exemplary embodiment, the length of theposition code 204 d is determined as follows,L≥log 4(N),  (1)wherein L represents the length of the position code 204 d (i.e., thenumber of nucleotides that make up the position code 204 d), and whereinN represents the total number of locations (x,y) to be encoded. As willbe described below, the library and/or library construction (such as thegeneration of the location-specific oligonucleotide sequence forpositional encoding) can optionally be provided as a service in a cloudenvironment.

According to an exemplary embodiment, the cDNA library is constructedusing MMLV RT. See, for example, FIG. 3 where an endogenous messengerRNA (mRNA) template 302 hybridizes with DNA oligonucleotide 204, andMMLV RT 304 synthesizes a DNA complement (cDNA) to the mRNA template302, and then appends a poly cytosine (C) sequence to the newlysynthesized cDNA sequence. For instance, as shown in step 310, in itssimplest form mRNA template 302 of the i^(th) gene includes a generic 5′CAP 302 a, a gene-specific coding region g_(i) 302 b, and a poly A tail302 c (i.e., a sequence of m adenine (A) repeats).

As shown in step 312, the mRNA template 302 hybridizes with the 3′ polyT sequence 204 e of DNA oligonucleotide 204, and the MMLV RT 304synthesizes a DNA complement (see for example gene-specific codingregion f_(i) 204 f) to the mRNA template 302. This new cDNA sequence isnow given reference numeral 204′. MMLV RT 304 then appends cDNA sequence204′ with poly C sequence 204 g.

According to an exemplary embodiment, a template switch is performedwhere a template switch oligonucleotide (TSO) sequence 402 is hybridizedwith the cDNA sequence 204′, after which the MMLV RT 304 performs the‘template switch’ in which MMLV RT 304 uses the TSO sequence 402 as atemplate for replication. See FIG. 4 . As shown in FIG. 4 , TSO sequence402 includes two groups, an oligonucleotide code 402 a that one wants toappend to the mRNA template 302, and a poly ribosomal Guanine (rG)repeat sequence 402 b. According to an exemplary embodiment, theoligonucleotide code 402 a is a PCR handle. As provided above, a PCRhandle enables PCR amplification.

As shown in step 410, the poly rG sequence 402 b of TSO 402 hybridizeswith the poly C sequence 204 g (that was appended to the cDNA sequence204′ by MMLV RT 304—see above). Doing so enables the MMLV RT 304 to thenuse TSO 402 as a template for replication. For instance, as shown instep 410 MMLV RT 304 appends a PCR handle 204 h to the poly C sequence204 g at the 3′ end of cDNA sequence 204′.

As shown in step 412, the cDNA sequence 204′ is then separated from themRNA template 302/TSO 402. By way of example only, the cDNA sequence204′ can be separated from the mRNA template 302/TSO 402 by ribonucleaseH activity of the MMLV RT technology and/or through the use of RNAdegradation by sodium hydroxide (NaOH) and heat. The result is amolecular probe with a unique oligonucleotide sequence (i.e., positioncode 204 d) that encodes positional data. For instance, as highlightedabove, each molecular probe is location-specific, meaning that itcontains an oligonucleotide sequence position code 204 d that is uniqueto a specific location of a biological sample. By way of the presenttechniques, the molecular probes are then delivered inside the cells atspecific locations of the biological sample corresponding to theoligonucleotide sequence position code 204 d each of molecular probecarries. As shown in step 414, the cDNA sequence 204′/molecular probescan be amplified by PCR.

As described in conjunction with the description of step 104 ofmethodology 100 above, the molecular probes with unique oligonucleotidesequences are then linked to a vessel such as a retrovirus, coupled todisulfide-linked cell-penetrating peptide (CPP) or bead micro-particlewhich will permit transfer of molecular probes into the cells atspecific locations of the biological sample. For live cells,retroviruses such as lentiviruses like the MMLV can be employed as thevessel. See FIG. 5 . Lentiviruses such as MMLV are advantageous as genedelivery vehicles because they are able to stably integrate into thegenome of cells. Further, among retroviruses, lentiviruses have thedistinguishing property of being able to insert genetic material intoboth dividing and non-dividing cells. The process for modifyingretroviruses such as lentiviruses for use as vectors for gene deliveryinto cells is well known to those of ordinary skill in the art.

A disulfide-linked cell-penetrating peptide (CPP) or activatablecell-penetrating peptide (ACCP) is also a suitable vessel fortransferring the molecular probes into the cells of the biologicalsample when the sample is live cells or tissue containing live cells.See FIG. 6 . CPP are biocarriers that are able to penetrate biologicalmembranes and thus translocate into cells, thereby permitting the cellsto internalize different cargo molecules. According to an exemplaryembodiment, the CPPs are short polycations attached viaprotease-cleavable linkers to neutralizing polyanions. Thus, as shown inFIG. 6 , the disulfide-linked CPP-to-oligonucleotides complexes arenon-permanent in the reducing environment within the cells. As such,once the CPP biocarriers deliver the molecular probes into theindividual cells of the biological sample, the disulfide bond betweenthe CPP biocarrier molecule and the molecular probe can be cleaved. Fora general description of CPP molecules as biocarriers see, for example,Gagat et al., “Cell-penetrating peptides and their utility in genomefunction modifications (Review),” International Journal of MolecularMedicine 40: 1615-1623 (October 2017), the contents of which areincorporated by reference as if fully set forth herein.

For tissue with living cells, bead micro-particles 702 are also asuitable vessel for transferring the molecular probes into the cells ofthe biological sample. See FIG. 7 . Beads micro-particles 702 are ableto permeate the biological membranes of cells by a process called beadtransfection. For instance, micro-particle beads such as glass beads canfirst be incubated in a solution containing the molecular probes. Themicro-particle beads now conjugated with molecular probes can then beintroduced into the cells using a process such as electroporation.According to an exemplary embodiment, the bead micro-particles 702 arethe same as bead 202 described above (see FIG. 2 ). Other vesseldelivery mechanisms (such as a retrovirus, coupled to a disulfide-linkedCPP, etc.) are needed because some mechanisms are better than others,depending on if they are being used for tissues or cells.

As described in conjunction with the description of step 106 ofmethodology 100 above, a liquid cargo delivery device such as amicrofluidic probe is employed to deliver the vessels with uniquemolecular probes to specific locations of the biological sample, wherethe vessel delivers the location-specific molecular probes inside thecells at those locations. See, for example, FIG. 8 . In step 810, aliquid cargo delivery device 802 (such as a microfluidic probe) scansthe surface of a biological sample 804 and deposits the vessels withunique molecular probes at specific locations (x,y) in the biologicalsample 804. A microfluidic probe is a non-contact, scanning platformthat can hydrodynamically localize as little as 100 picoliters of aliquid cargo with micrometer precision.

By way of example only, the liquid cargo delivery device 802 dispenses acontrolled amount of a processing solution (e.g., an aqueous solution)containing the vessels/molecular probes at multiple locations (i.e.,(x₁,y₁), (x₁,y₂), (x₁,y₃), etc.) in the biological sample 804. See step812. As provided above, once the vessel/molecular probe is delivered toa specific location of the biological sample 804, the vessel deliversthe molecular probes inside the cell(s) 806 at that specific location.

After the location-specific molecular probes have beendelivered/inserted into the cells 806, the cells 806 are extracted fromthe biological sample 804. See step 814. However, even after beingdisassociated from the biological sample 804, the individual cells 806retain the molecular probe with oligonucleotide sequence encoding theoriginal position of the cells 806 in the biological sample 804. Thus,this positional data can be retained through the subsequent sequencingprocess. See step 816.

For example, one or more single cell sequencing techniques can beperformed. Suitable single cell sequencing techniques include, but arenot limited to, drop-seq, seq-well, cyto-seq, and combinations thereof.The single cell sequencing performed in step 816 can be used to identifythe subject cell by the cell barcode (see above), the original positionof the cells 806 within the biological sample 804 via the unique,location-specific oligonucleotide sequence of the molecular probes,and/or transcriptome information of the cells 806. Therefore, thecombination of the present positional delivery and encoding process withextraction and single cell sequencing can collect concomitant spatialand molecular measurements (e.g., position coordinates andtranscriptomes of one or more of the cells 806 in the biological sample804) which, as described in conjunction with the description of step 110of methodology 100 above, can be recorded and/or analyzed in silico.

The present invention may be a system, a method, and/or a computerprogram product. The computer program product may include a computerreadable storage medium (or media) having computer readable programinstructions thereon for causing a processor to carry out aspects of thepresent invention.

The computer readable storage medium can be a tangible device that canretain and store instructions for use by an instruction executiondevice. The computer readable storage medium may be, for example, but isnot limited to, an electronic storage device, a magnetic storage device,an optical storage device, an electromagnetic storage device, asemiconductor storage device, or any suitable combination of theforegoing. A non-exhaustive list of more specific examples of thecomputer readable storage medium includes the following: a portablecomputer diskette, a hard disk, a random access memory (RAM), aread-only memory (ROM), an erasable programmable read-only memory (EPROMor Flash memory), a static random access memory (SRAM), a portablecompact disc read-only memory (CD-ROM), a digital versatile disk (DVD),a memory stick, a floppy disk, a mechanically encoded device such aspunch-cards or raised structures in a groove having instructionsrecorded thereon, and any suitable combination of the foregoing. Acomputer readable storage medium, as used herein, is not to be construedas being transitory signals per se, such as radio waves or other freelypropagating electromagnetic waves, electromagnetic waves propagatingthrough a waveguide or other transmission media (e.g., light pulsespassing through a fiber-optic cable), or electrical signals transmittedthrough a wire.

Computer readable program instructions described herein can bedownloaded to respective computing/processing devices from a computerreadable storage medium or to an external computer or external storagedevice via a network, for example, the Internet, a local area network, awide area network and/or a wireless network. The network may comprisecopper transmission cables, optical transmission fibers, wirelesstransmission, routers, firewalls, switches, gateway computers and/oredge servers. A network adapter card or network interface in eachcomputing/processing device receives computer readable programinstructions from the network and forwards the computer readable programinstructions for storage in a computer readable storage medium withinthe respective computing/processing device.

Computer readable program instructions for carrying out operations ofthe present invention may be assembler instructions,instruction-set-architecture (ISA) instructions, machine instructions,machine dependent instructions, microcode, firmware instructions,state-setting data, or either source code or object code written in anycombination of one or more programming languages, including an objectoriented programming language such as Smalltalk, C++ or the like, andconventional procedural programming languages, such as the “C”programming language or similar programming languages. The computerreadable program instructions may execute entirely on the user'scomputer, partly on the user's computer, as a stand-alone softwarepackage, partly on the user's computer and partly on a remote computeror entirely on the remote computer or server. In the latter scenario,the remote computer may be connected to the user's computer through anytype of network, including a local area network (LAN) or a wide areanetwork (WAN), or the connection may be made to an external computer(for example, through the Internet using an Internet Service Provider).In some embodiments, electronic circuitry including, for example,programmable logic circuitry, field-programmable gate arrays (FPGA), orprogrammable logic arrays (PLA) may execute the computer readableprogram instructions by utilizing state information of the computerreadable program instructions to personalize the electronic circuitry,in order to perform aspects of the present invention.

Aspects of the present invention are described herein with reference toflowchart illustrations and/or block diagrams of methods, apparatus(systems), and computer program products according to embodiments of theinvention. It will be understood that each block of the flowchartillustrations and/or block diagrams, and combinations of blocks in theflowchart illustrations and/or block diagrams, can be implemented bycomputer readable program instructions.

These computer readable program instructions may be provided to aprocessor of a general purpose computer, special purpose computer, orother programmable data processing apparatus to produce a machine, suchthat the instructions, which execute via the processor of the computeror other programmable data processing apparatus, create means forimplementing the functions/acts specified in the flowchart and/or blockdiagram block or blocks. These computer readable program instructionsmay also be stored in a computer readable storage medium that can directa computer, a programmable data processing apparatus, and/or otherdevices to function in a particular manner, such that the computerreadable storage medium having instructions stored therein comprises anarticle of manufacture including instructions which implement aspects ofthe function/act specified in the flowchart and/or block diagram blockor blocks.

The computer readable program instructions may also be loaded onto acomputer, other programmable data processing apparatus, or other deviceto cause a series of operational steps to be performed on the computer,other programmable apparatus or other device to produce a computerimplemented process, such that the instructions which execute on thecomputer, other programmable apparatus, or other device implement thefunctions/acts specified in the flowchart and/or block diagram block orblocks.

The flowchart and block diagrams in the Figures illustrate thearchitecture, functionality, and operation of possible implementationsof systems, methods, and computer program products according to variousembodiments of the present invention. In this regard, each block in theflowchart or block diagrams may represent a module, segment, or portionof instructions, which comprises one or more executable instructions forimplementing the specified logical function(s). In some alternativeimplementations, the functions noted in the block may occur out of theorder noted in the figures. For example, two blocks shown in successionmay, in fact, be executed substantially concurrently, or the blocks maysometimes be executed in the reverse order, depending upon thefunctionality involved. It will also be noted that each block of theblock diagrams and/or flowchart illustration, and combinations of blocksin the block diagrams and/or flowchart illustration, can be implementedby special purpose hardware-based systems that perform the specifiedfunctions or acts or carry out combinations of special purpose hardwareand computer instructions.

Turning now to FIG. 9 , a block diagram is shown of an apparatus 900 forimplementing one or more of the methodologies presented herein. By wayof example only, apparatus 900 can be configured to implement one ormore of the steps of methodology 100 of FIG. 1 . For instance, accordingto an exemplary embodiment, apparatus 900 may be configured to storeand/or analyze the transcriptomic data extracted from the single cellsequencing along with the unique positional data obtained from themolecular probes indicating the original positioning of the cells in thebiological sample.

Apparatus 900 includes a computer system 910 and removable media 950.Computer system 910 includes a processor device 920, a network interface925, a memory 930, a media interface 935 and an optional display 940.Network interface 925 allows computer system 910 to connect to anetwork, while media interface 935 allows computer system 910 tointeract with media, such as a hard drive or removable media 950.

Processor device 920 can be configured to implement the methods, steps,and functions disclosed herein. The memory 930 could be distributed orlocal and the processor device 920 could be distributed or singular. Thememory 930 could be implemented as an electrical, magnetic or opticalmemory, or any combination of these or other types of storage devices.Moreover, the term “memory” should be construed broadly enough toencompass any information able to be read from, or written to, anaddress in the addressable space accessed by processor device 920. Withthis definition, information on a network, accessible through networkinterface 925, is still within memory 930 because the processor device920 can retrieve the information from the network. It should be notedthat each distributed processor that makes up processor device 920generally contains its own addressable memory space. It should also benoted that some or all of computer system 910 can be incorporated intoan application-specific or general-use integrated circuit.

Optional display 940 is any type of display suitable for interactingwith a human user of apparatus 900. Generally, display 940 is a computermonitor or other similar display.

Referring to FIG. 10 and FIG. 11 , it is to be understood that althoughthis disclosure includes a detailed description on cloud computing,implementation of the teachings recited herein are not limited to acloud computing environment. Rather, embodiments of the presentinvention are capable of being implemented in conjunction with any othertype of computing environment now known or later developed.

Cloud computing is a model of service delivery for enabling convenient,on-demand network access to a shared pool of configurable computingresources (e.g., networks, network bandwidth, servers, processing,memory, storage, applications, virtual machines, and services) that canbe rapidly provisioned and released with minimal management effort orinteraction with a provider of the service. This cloud model may includeat least five characteristics, at least three service models, and atleast four deployment models.

Characteristics are as follows:

On-demand self-service: a cloud consumer can unilaterally provisioncomputing capabilities, such as server time and network storage, asneeded automatically without requiring human interaction with theservice's provider.

Broad network access: capabilities are available over a network andaccessed through standard mechanisms that promote use by heterogeneousthin or thick client platforms (e.g., mobile phones, laptops, and PDAs).

Resource pooling: the provider's computing resources are pooled to servemultiple consumers using a multi-tenant model, with different physicaland virtual resources dynamically assigned and reassigned according todemand. There is a sense of location independence in that the consumergenerally has no control or knowledge over the exact location of theprovided resources but may be able to specify location at a higher levelof abstraction (e.g., country, state, or datacenter).

Rapid elasticity: capabilities can be rapidly and elasticallyprovisioned, in some cases automatically, to quickly scale out andrapidly released to quickly scale in. To the consumer, the capabilitiesavailable for provisioning often appear to be unlimited and can bepurchased in any quantity at any time.

Measured service: cloud systems automatically control and optimizeresource use by leveraging a metering capability at some level ofabstraction appropriate to the type of service (e.g., storage,processing, bandwidth, and active user accounts). Resource usage can bemonitored, controlled, and reported, providing transparency for both theprovider and consumer of the utilized service.

Service Models are as follows:

Software as a Service (SaaS): the capability provided to the consumer isto use the provider's applications running on a cloud infrastructure.The applications are accessible from various client devices through athin client interface such as a web browser (e.g., web-based e-mail).The consumer does not manage or control the underlying cloudinfrastructure including network, servers, operating systems, storage,or even individual application capabilities, with the possible exceptionof limited user-specific application configuration settings.

Platform as a Service (PaaS): the capability provided to the consumer isto deploy onto the cloud infrastructure consumer-created or acquiredapplications created using programming languages and tools supported bythe provider. The consumer does not manage or control the underlyingcloud infrastructure including networks, servers, operating systems, orstorage, but has control over the deployed applications and possiblyapplication hosting environment configurations.

Infrastructure as a Service (IaaS): the capability provided to theconsumer is to provision processing, storage, networks, and otherfundamental computing resources where the consumer is able to deploy andrun arbitrary software, which can include operating systems andapplications. The consumer does not manage or control the underlyingcloud infrastructure but has control over operating systems, storage,deployed applications, and possibly limited control of select networkingcomponents (e.g., host firewalls).

Deployment Models are as follows:

Private cloud: the cloud infrastructure is operated solely for anorganization. It may be managed by the organization or a third party andmay exist on-premises or off-premises.

Community cloud: the cloud infrastructure is shared by severalorganizations and supports a specific community that has shared concerns(e.g., mission, security requirements, policy, and complianceconsiderations). It may be managed by the organizations or a third partyand may exist on-premises or off-premises.

Public cloud: the cloud infrastructure is made available to the generalpublic or a large industry group and is owned by an organization sellingcloud services.

Hybrid cloud: the cloud infrastructure is a composition of two or moreclouds (private, community, or public) that remain unique entities butare bound together by standardized or proprietary technology thatenables data and application portability (e.g., cloud bursting forload-balancing between clouds).

A cloud computing environment is service oriented with a focus onstatelessness, low coupling, modularity, and semantic interoperability.At the heart of cloud computing is an infrastructure that includes anetwork of interconnected nodes.

Referring now to FIG. 10 , illustrative cloud computing environment 50is depicted. As shown, cloud computing environment 50 includes one ormore cloud computing nodes 10 with which local computing devices used bycloud consumers, such as, for example, personal digital assistant (PDA)or cellular telephone 54A, desktop computer 54B, laptop computer 54C,and/or automobile computer system 54N may communicate. Nodes 10 maycommunicate with one another. They may be grouped (not shown) physicallyor virtually, in one or more networks, such as Private, Community,Public, or Hybrid clouds as described hereinabove, or a combinationthereof. This allows cloud computing environment 50 to offerinfrastructure, platforms and/or software as services for which a cloudconsumer does not need to maintain resources on a local computingdevice. It is understood that the types of computing devices 54A-N shownin FIG. 10 are intended to be illustrative only and that computing nodes10 and cloud computing environment 50 can communicate with any type ofcomputerized device over any type of network and/or network addressableconnection (e.g., using a web browser).

Referring now to FIG. 11 , a set of functional abstraction layersprovided by cloud computing environment 50 (FIG. 10 ) is shown. Itshould be understood in advance that the components, layers, andfunctions shown in FIG. 11 are intended to be illustrative only andembodiments of the invention are not limited thereto. As depicted, thefollowing layers and corresponding functions are provided:

Hardware and software layer 60 includes hardware and softwarecomponents. Examples of hardware components include: mainframes 61; RISC(Reduced Instruction Set Computer) architecture based servers 62;servers 63; blade servers 64; storage devices 65; and networks andnetworking components 66. In some embodiments, software componentsinclude network application server software 67 and database software 68.

Virtualization layer 70 provides an abstraction layer from which thefollowing examples of virtual entities may be provided: virtual servers71; virtual storage 72; virtual networks 73, including virtual privatenetworks; virtual applications and operating systems 74; and virtualclients 75.

In one example, management layer 80 may provide the functions describedbelow. Resource provisioning 81 provides dynamic procurement ofcomputing resources and other resources that are utilized to performtasks within the cloud computing environment. Metering and Pricing 82provide cost tracking as resources are utilized within the cloudcomputing environment, and billing or invoicing for consumption of theseresources. In one example, these resources may include applicationsoftware licenses. Security provides identity verification for cloudconsumers and tasks, as well as protection for data and other resources.User portal 83 provides access to the cloud computing environment forconsumers and system administrators. Service level management 84provides cloud computing resource allocation and management such thatrequired service levels are met. Service Level Agreement (SLA) planningand fulfillment 85 provide pre-arrangement for, and procurement of,cloud computing resources for which a future requirement is anticipatedin accordance with an SLA.

Workloads layer 90 provides examples of functionality for which thecloud computing environment may be utilized. Examples of workloads andfunctions which may be provided from this layer include: mapping andnavigation 91; software development and lifecycle management 92; virtualclassroom education delivery 93; data analytics processing 94;transaction processing 95; and cDNA library construction 96.

Although illustrative embodiments of the present invention have beendescribed herein, it is to be understood that the invention is notlimited to those precise embodiments, and that various other changes andmodifications may be made by one skilled in the art without departingfrom the scope of the invention.

What is claimed is:
 1. A method, comprising: encoding cells in abiological sample for single cell sequencing by delivering molecularprobes inside the cells that encode positional data comprising aposition of the cells in the biological sample such that positional datais retained, via the molecular probes, even when the cells aredisassociated from the biological sample for the single cell sequencing.2. The method of claim 1, wherein the molecular probes comprise anoligonucleotide sequence that uniquely encodes the position of the cellsin the biological sample.
 3. The method of claim 2, wherein themolecular probes further comprise: a polymerase chain reaction (PCR)handle; a cell barcode; and a unique molecular identifier (UMI).
 4. Themethod of claim 1, wherein the biological sample is selected from thegroup consisting of: a cell culture, a tissue sample, and combinationsthereof.
 5. The method of claim 1, further comprising: constructing acomplementary deoxyribonucleic acid (cDNA) library of the molecularprobes.
 6. The method of claim 5, wherein the cDNA library isconstructed using Moloney murine leukemia virus reverse transcriptase(“MMLV RT”) technology.
 7. The method of claim 1, wherein the molecularprobes are delivered inside the cells using a vessel selected from thegroup consisting of: a retrovirus, cell-penetrating peptides, and beadmicroparticles.
 8. The method of claim 7, wherein the vessel comprises aretrovirus, and wherein the retrovirus is a lentivirus.
 9. The method ofclaim 7, wherein the vessel comprises cell-penetrating peptides, andwherein the cell-penetrating peptides comprise cleavabledisulfide-linked cell-penetrating peptides.
 10. The method of claim 7,wherein the vessel comprises bead microparticles, and wherein the beadmicroparticles comprise glass beads.
 11. The method of claim 7, furthercomprising: linking the molecular probes to the vessel; and deliveringthe vessel with the molecular probes to specific locations of thebiological sample where the vessel delivers the molecular probes insidethe cells at the specific locations.
 12. The method of claim 11, whereinthe vessel with the molecular probes are delivered to the specificlocations of the biological sample using a liquid cargo delivery device.13. The method of claim 12, wherein the liquid cargo device comprises amicrofluidic probe.
 14. The method of claim 1, further comprising:extracting the cells containing the molecular probes from the biologicalsample; and performing single cell sequencing of the extracted cells.15. A method, comprising: constructing a cDNA library of molecularprobes that encode a position of cells in a biological sample; linkingthe molecular probes to a vessel; delivering the vessel with themolecular probes to specific locations of the biological sample wherethe vessel delivers the molecular probes inside the cells at thespecific locations; extracting the cells containing the molecular probesfrom the biological sample, wherein the molecular probes retainpositional data of the cells in the biological sample following theextracting; and performing single cell sequencing of the extractedcells.
 16. The method of claim 15, wherein the molecular probes comprisean oligonucleotide sequence that uniquely encodes the position of thecells in the biological sample.
 17. The method of claim 15, wherein thevessel is selected from the group consisting of: a retrovirus,cell-penetrating peptides, and bead microparticles.
 18. The method ofclaim 15, wherein the vessel with the molecular probes are delivered tothe specific locations of the biological sample using a liquid cargodelivery device.
 19. The method of claim 18, wherein the liquid cargodevice comprises a microfluidic probe.